Retroviral vector, a replication system for said vector and avian or mammalian cells transfected with said vector

ABSTRACT

A retroviral vector comprising a retrovirus with infectivity for birds and/or mammals in which at least part of the genomic RNA sequences carrying information for the production of viral proteins required in trans for retroviral replication have been replaced by one or more sequences carrying information to be introduced in a target cell chromosome, characterized in that the primer binding site (PBS) has been modified to a sequence that does not allow strong base pairing with the 3&#39; end or other priming sequence in any naturally occurring tRNA.

The present application is a continuation of our prior-filed applicationSer. No. 08/525,849, filed Sep. 8, 1995, now U.S. Pat. No. 5,866,411.

This invention concerns a retroviral vector comprising a retrovirus withinfectivity for birds and mammals, in which at least part of the genomicRNA sequences carrying information for the production of viral proteinsrequired in trans for retroviral replication have been replaced by oneor more sequences carrying information to be introduced in a target cellchromosome. The invention also concerns a tRNA-like primer for reversetranscription of said retroviral vector, a DNA sequence comprising thestructural gene sequence of the tRNA-like primer, and a packaging cellline for viral replication of said retroviral vector. Further, theinvention concerns an avian or mammalian cell which has been transfectedwith said retroviral vector.

BACKGROUND OF THE INVENTION

Retroviral vectors are gene transfer vehicles for birds and mammals thatexploit features of the retrovirus replication cycle such as highinfection efficiency and stable co-linear integration of the virallytransmitted information in a target cell chromosome. Retroviral vectorsare becoming important tools in basic research, biotechnology and genetherapy.

Most retroviral vectors currently in use are derived from MurineLeukemia Viruses (MLVs). MLVs are particularly suitable as vectors dueto their well-documented pattern of transcription in diverse cell typesand relatively simple modular genetic structure.

1. Retroviral structure

Retroviruses belong among the enveloped viruses (FIG. 1). The bilipidenvelope is derived from the host cell membrane and modified by theinsertion of the viral surface protein (SU) and transmembrane protein(TM). The matrix protein (MA) is situated just under the outer membranesurrounding the inner core. The core consists of capsid protein (CA).Inside the capsid are two copies of the retroviral genome which areattached to each other at the 5' end via hydrogen bonding. The viruscore particle also contains the viral enzymes: reverse transcriptase(RT), protease (PR), and integrase (IN), and the nucleocapsid protein(NC) which is bound to the viral genome. Besides these proteins encodedby the virus, the virion also contains a number of tRNA moleculesderived from the host cell tRNA population.

Murine leukemia viruses (MLV) belong to the simple retroviruses.Retroviruses have a characteristic genomic map: Two long terminalrepeats (LTRs) flanking the three structural genes gag, pol and env(FIG. 2). The LTRs can be subdivided into three regions: The U3 regioncontaining the enhancer and promoter elements recognized by the cellulartranscription machinery, the R region which play an important roleduring reverse transcription and furthermore contains thepolyadenylation signal, and the U5 region containing sequences ofimportance in reverse transcription and packaging of the retroviralgenome. Additionally, the LTRs contain cis elements, the invertedrepeats, important during the process of integration (FIG. 3).

The integrated provirus gives rise to two mRNA transcripts, afull-length mRNA encoding the gag-, and the gag-pol polyproteins, and aspliced mRNA encoding the envelope glycoproteins. The full-length mRNAalso serves as the genomic RNA and, besides the already describedcomponents of the LTR moieties, contains three important cis elements inthe 5' untranslated sequence. The primer binding site (PBS), situateddownstream from the U5 region, consists of 18 nucleotides complementaryto the 3' end of the primer tRNA molecule. Also located in the 5'untranslated region, between the PBS and the beginning of the gag openreading frame, is the packaging signal (Ψ). The 5' untranslated regionfurthermore contains a dimer linkage domain responsible for thedimerization of the two viral genomes in the virion. Immediatelyupstream from the U3 region is another important cis-element, thepolypurine tract (PP), which consists of a stretch of A and G residues.This element serves as a site for priming plus-strand DNA synthesisduring reverse transcription.

2. The retroviral lifecycle

The retroviral lifecycle is outlined in FIG. 4. Two different mechanismshave been proposed to explain the entry of the virus particle into thehost cytoplasm. Most retroviruses, including MLV, are thought to enterthe host cell through receptor-mediated endocytosis, a process in whichthe whole enveloped virus particle is internalized into an endosomalbody. The receptor molecule for the ecotropic murine leukemia viruseshas been cloned and identified as a cationic amino acid transporter.

After the viral core particle has entered the cytoplasm of the hostcell, all enzymatic functions leading to the integrated double-strandedDNA provirus are managed by viral proteins synthesized in the previoushost cell and brought along in the virion. The fate of the viralproteins after entry of the core particle is not clear, but the reversetranscriptase, the integrase and the capsid protein remain with the RNAgenome forming the nucleoprotein complex in which reverse transcriptionoccurs. Recently, also the matrix protein has been found in associationwith the nucleoprotein complex.

Following reverse transcription, the nucleoprotein complex migrates tothe host cell nucleus. The mechanism responsible for the nuclearlocalization is unclear, although evidence from Rous sarcoma virus (RSV)suggests the IN protein to be important since the RSV IN protein, whenproduced in mammalian cells, is localized in the nucleus. Entry of thenucleoprotein complex into the nucleus requires mitosis, probablybecause the nucleoprotein complex cannot penetrate the nuclear envelope.Once in the nucleus, integration is mediated by the IN protein. The INprotein recognizes the conserved inverted repeats at the ends of theLTRs and removes 2 bases from the 3' hydroxyl termini of both strands.The IN protein also catalyzes a cleavage in the host DNA and mediatesthe connections between the proviral DNA and the host DNA. As for thespecificity of integration no consensus host DNA target sequence hasbeen found, although a tendency to integrate near DNase I-hypersensitivesites has been reported.

For the simple retroviruses (including MLV) transcription andtranslation is performed by the host cell machinery. Complex viruses(including HIV and HTLV) encode transactivating proteins involved intranscriptional regulation. The assembly of MLV particles takes place atthe host membrane, and the process coincides with the budding process.In mammalian B and C type viruses (MMTV and HTLV, respectively) viralcore particles are assembled in the host cell cytoplasm. Encapsidationof the viral genomic RNA is mediated through binding of the cis-actingencapsidation signal and the NC moiety of the Gag- or the Gag-Polprecursor protein.

After budding, the Gag- and Gag-Pol polyproteins are cleaved by viralprotease (PR). Maturation of the viral proteins results in an overallchange in virion morphology. In addition to proteolytic cleavage of theviral polyproteins following budding, the genomic RNA also undergoes amaturation process resulting in a compact dimeric genome.

3. Reverse transcription

The enzyme reverse transcriptase was discovered in 1970, and the currentmodel of reverse transcription was proposed in 1979 (Gilboa et al.1979). In addition to a DNA polymerase that can copy either RNA or DNAtemplates, reverse transcriptase contains an RNase H which selectivelydegrades RNA in RNA/DNA hybrids.

Retroviruses utilize a cellular tRNA molecule as a primer during reversetranscription. Different retroviruses utilize different tRNA species asprimers in reverse transcription. Murine leukemia viruses (MLVs) andhuman T-cell leukemia virus (HTLV) use a proline-tRNA primer, humanimmunodeficiency virus (HIV) and mouse mammary tumor virus (MMTV)utilize a lysine-tRNA primer, whereas avian leukosis sarcoma virus(ALSV) use a tryptophan-tRNA molecule as a primer for reversetranscription. However, within a given group of retroviruses, thesequence of the primer binding site is highly conserved duringretroviral replication, where the 3' 18 nucleotides of the tRNA moleculeare copied during plus-strand synthesis. Furthermore, during the virallife cycle the tRNA primer is likely to specifically interact with viralproteins and the viral genome during the processes of packaging,annealing, and reverse transcription. Specific interactions between thetRNA primer and the viral proteins reverse transcriptase and thenucleocapsid protein, as well a secondary interactions between the tRNAand viral genomic RNA have been demonstrated in several viruses.

In a recent article (Lund et al. 1993) the present inventors havealtered the MLV wild-type PBS matching a proline-tRNA to sequencesmatching either a glutamine- or a lysine-tRNA. The effect of the alteredPBS sequences was studied by single cycle replication of a retroviralvector, enabling them to quantify the effect of the introducedmutations. The structure of the transduced PBSs was analyzed byamplification of proviral vectors followed by direct sequencing of thePBS and surrounding DNA. They found that MLV can replicate by usingvarious tRNA molecules as primers and concluded that primer bindingsite-tRNA primer interactions are of major importance for tRNA primerselection, but that efficient primer selection does not require perfectWatson-Crick base pairing at all 18 positions of the primer bindingsite. Later they have proved that MLV can also replicate by using themethionine (initiator)-tRNA as the primer.

The tRNA primer is packaged in the virion during virus assembly, andoriginates therefore from the previous host cell. The tRNA primermolecule interacts through base pairing with a region of the genomic RNA(the primer binding site) situated downstream from the U5 region at the5' end of the genome (FIG. 5). In murine leukemia viruses 18 bases fromthe aminoacceptor stem and the TΨC loop of the tRNA are annealed to thePBS and the reverse transcription of the retroviral genome is initiatedfrom the 3' end of the tRNA molecule. From here reverse transcriptasesynthesizes the first DNA, the minus-strand strong stop DNA. The 3' endof the minus-strand strong stop DNA contains a copy of the R regioncomplementary to the 3' R region of the genomic RNA. The newlysynthesized DNA is thought to relocate to this region (first jump) fromwhere minus-strand DNA synthesis can proceed. As the growingminus-strand is synthesized, RNase H continues to degrade the viral RNA.A fragment of the viral RNA, situated at the polypurine tract (PP), isleft and acts as the primer for plus-strand DNA synthesis. Plus-strandDNA synthesis proceeds through U3, R and U5 and copies the bases of thetRNA molecule that are complementary to the PBS. At position 57 in thetRNA molecule the reverse transcriptase encounters a modified nucleotide(m¹ A), which it presumably cannot use as a template, and synthesis ofthe plus-strand terminates. RNase H removes the overhanging part of thetRNA molecule. The remaining part of the plus-strand, leading to thecomplete double-stranded DNA provirus, is primed by the first part aftera relocation event where the two complementary copies of the PBSinteract through base pairing. Resulting from this complex reaction is adouble-stranded DNA provirus, which is longer than the RNA template dueto the copying of repetitive sequences U3 and U5.

4. Retroviral vector systems

A central element in this invention is a retroviral vector propagationsystem in which infectious, recombinant virus particles can be producedwithout contamination with replication competent wild type virus. Themain advantage of retroviral vectors is the utilization of the efficientinfection and integration processes developed in the viruses throughevolution.

The proviral genome of MLV can be divided into sequences that arerequired in cis or in trans for viral replication (FIG. 6). Thecis-acting elements are located at the ends of the proviral genome andencompass the U3, R and U5 regions, the inverted repeats in U3 and U5,the PBS, the polypurine tract and the packaging signal. These sequencescontain all the elements needed for correct reverse transcription andintegration and make up the minimal requirements for a retroviralvector. The space needed for the insertion of foreign DNA is created bydeleting the sequences encompassing the open reading frames gag, pol andenv.

For most applications it is essential that the retroviral vectors usedfor gene transfer are unable to generate new virus progeny in the targetcell. This requires that the vector constructs are replicationdefective, the components required to complete the life cycle beingsupplied from loci outside the vector construct in the virus producingcell.

If these components are not present in the target cell, further virusgeneration is not possible. However, replication competent viruses mayarise as a result of recombination. Such virus may eventually havepathological consequences including the malignant transformation of theinfected target cell.

Retroviral vectors are normally propagated by the use of specializedpackaging cell lines. In such packaging cell lines all trans-actingvirus-encoded components are produced from loci outside the vectorconstruct.

The first packaging cell line was constructed by Mann et al. (1983).This cell line (Ψ-2), containing a Moloney-MLV proviral genome with a350 bp deletion overlapping the packaging signal, produces all the viralproteins needed in trans for virion production (FIG. 7A). Aftertransfection into this cell line, retroviral vectors carrying thenecessary cis-signals will be packaged into infectious virions (FIG.7B). Although the early packaging cell lines Ψ-2 (Mann et al. 1983) andΨ-AM¹ (Cone & Mulligan 1984) have been used successfully in a largenumber of studies, they suffer from important deficiencies. Despite thedeletion of the packaging signal replication competent viruses mayarise, presumably as a result of recombination between the packagingconstruct and introduced vector sequences or viruses endogenous to thegenome of the packaging cell (Miller 1990). Recombination occurs at highfrequency in retroviral replication. The rate of homologousrecombination between two heterologous genomes packaged in the samevirion has been estimated to range as high as 10 to 30 % for each roundof replication. A number of studies have reported recombination betweenintroduced viruses or vectors and homologous sequences endogenous to thehost cell. Further evidence that deletion of the packaging signal is notsufficient to prevent packaging, comes from a study of a retroviralvector without a packaging signal where packaging and transfer of thisvector was easily detected, even though the transduction rate was 3000fold lower than that of a vector containing the packaging signal.Obviously, the 350 bp deletion in the 5' untranslated region does notinhibit packaging, a fact supported by reports indicating that sequenceswithin the gag open reading frame and within the U5 region also may playa role in the encapsidation process. Furthermore, retroviral particlesalso package non-viral RNA, indicating that both specific recognitionand a more general affinity for RNA is involved in the encapsidationprocess. Moreover, only a single recombination event is needed torestore the packaging signal and regenerate replication competentviruses. There are several reports of helper virus regenerationresulting from homologous recombination between the packaging constructand either introduced vectors or endogenous viral sequences.

¹ The Ψ-AM packaging construct resembles Ψ-2 but expresses another envgene giving rise to recombinant viruses with an amphotropic host range.

To further reduce the risk of generation of complete viruses byrecombination in the packaging cells, second generation pakcaging celllines have been constructed. The main advantage of these new packagingcell lines is that the packaging construct has been divided into twoseparate constructs; one encoding the gag-pol polyprotein and onecarrying the env gene (FIG. 8). By splitting the packaging construct therisk of recombination is greatly reduced (Miller 1990). By reducing thesequence homology of the packaging construct to the introduced vectorsand endogenous MLV-like sequences the risk of homologous recombinationcan be further reduced.

However, not only the engineered parts of the packaging cells but alsoendogenous retroviral sequences in the DNA of the packaging cell as wellas in the target cell may have to be considered in safety assessments.The risk of a contribution from endogenous retroviruses may be reducedby use of packaging cells based upon cell lines from other species wherethe endogenous retroviral elements may be more divergent. The currentpackaging cell technology has recently been reviewed (D. Valerio 1992).

A recent publication (Chapman et al. 1992) describes that the yeastretrotransposon Tyl uses tRNA_(i) ^(Met) as a primer for Tyl reversetranscription, and that mutations in the Tyl element that alter 5 of the10 nucleotides that are complementary to the tRNA_(i) ^(Met) abolish Tyltransposition. When a yeast strain is constructed which lacks wild-typetRNA_(i) ^(Met) and is dependent on a mutant derivative of tRNA_(i)^(Met) that has an altered acceptor stem sequence, engineered to restorehomology with the Tyl-PBS mutant, the compensatory mutations made in thetRNA_(i) ^(Met) alleviate the transposition defect of the Tyl-PBSmutant. The mutant and wild-type tRNA_(i) ^(Met) are enriched within Tylvirus-like particles irrespective of complementarity to the Tyl-PBS, andfrom this the authors conclude that complementarity between the Tyl-PBSand tRNA_(i) ^(Met) is essential for transposition, but is not necessaryfor packaging of the tRNA inside virus-like particles.

SUMMARY OF THE INVENTION

To further reduce the risk of uncontrolled regeneration of completevirus or for other means of vector spread resulting from an interactionof a retroviral vector with various engineered or endogenous cis- ortrans-acting components, the present invention proposes to make thevector transfer dependent upon a specifically engineered, tRNA-likeprimer for reverse transcription. Thus, in principle, only specializedpackaging cells provided with an appropriate artificial primer wouldallow vector propagation (FIG. 9).

Another aspect of this invention is the potential use of retroviralvectors as antiviral agents. Such vectors may be directed againstpathogenic viruses related to the type used for construction of thevector, thus having the same host range and cellular tropism. Thisstrategy may turn out to be of particular importance for antiviralapproaches towards HIV or HTLV-1. A key point in this strategy is todirect antiviral activity against critical cis-acting genetic regions ofthe pathogenic virus e.g. by antisense or ribozyme constructs. Theprimer binding site (PBS) is an important and conserved cis region andtherefore constitutes a possible target site. Retroviral vectorsutilizing an artificial reverse transcription primer and hence carryinga modified PBS would be particularly advantageous, since the antiviralactivity of such vectors would not be directed towards their own ciselements.

As mentioned above, all retroviruses require a specific tRNA primer forreverse transcription; however different groups of retroviruses may usedifferent tRNA species. The tRNA-primer usually interacts with acomplementary sequence, the primer binding site (PBS) near the 5' end ofthe viral genomic RNA. We have addressed the possibility of using anartificial tRNA-like primer for transmission of vectors derived frommurine leukemia viruses (MLVs). Most available retroviral systems werederived from this group of viruses and all approved clinical protocolsfor gene therapy or gene marking by retroviral vectors use vectors basedupon this specific group. The tRNA utilized as a primer for reversetranscription by MLV is a proline-specific tRNA (tRNA^(pro1) ortRNA^(pro2)) that interacts with a complementary 18 nucleotidesPBS-sequence in the retroviral genomic RNA.

Accordingly, the present invention provides a retroviral vectorcomprising a retrovirus with infectivity for birds and/or mammals inwhich at least part of the genomic RNA sequences carrying informationfor the production of viral proteins required in trans for retroviralreplication have been replaced by one or more sequences carryinginformation to be introduced in a target cell chromosome, saidretroviral vector being characterized in that the primer binding site(PBS) has been modified to a sequence that does not allow strong basepairing with the 3' end or other priming sequence in any naturallyoccurring tRNA.

By "strong base pairing" is meant a degree of binding that allowspriming of a complementary DNA strand in the reverse transcription ofthe vector RNA. A sequence that does not allow strong base pairingshould preferably show less than 50% homology, and more preferably lessthan 33% homology, with the 3' end or other priming sequence in anynaturally occurring tRNA.

A suitable retroviral vector according to the invention is derived frommurine leukemia virus (MLV) as described above; and in a specific vectorof this kind the primer binding site (PBS) has been modified to thesequence stated in the following SEQ ID No. 1 UGGGAUGAAUCUAGGGAU.

To further reduce the possibility of binding of naturally occurring tRNAmolecules to modified primer binding sites it may be advantageous toalter the three 5' nucleotides (UGG) of the PBS thereby removing anycomplementarity to the 3' CCA-tail of naturally occurring tRNAmolecules.

In a retroviral vector according to the invention, especially in onederived from murine leukemia virus (MLV), it may be advantageous also tomodify up to 5 nucleotides 3' of the primer binding site to a sequencethat does not allow additional base pairing with a tRNA primer.

The invention also provides a replication system for the retroviralvector according to the invention, said system comprising a tRNA-likeprimer for reverse transcription of the retroviral vector as well as aDNA sequence comprising the structural gene sequence of said primerwhich is incorporated in a packaging cell line for viral replication ofthe retroviral vector.

A tRNA-like primer according to the invention is derived from a tRNApriming the reverse transcription of the retrovirus, from which thevector is derived, by modifying the 3' end or other sequence thereof,which is to be active in priming, to a sequence that allows strongbase-pairing with the PBS of the vector.

In such a tRNA-like primer it may be necessary or advantageous that alsosequences involved in forming the secondary clover-leaf structure of thetRNA molecule by internal base-pairing with the modified sequences aremodified to be complementary therewith.

If the three 5' nucleotides in the PBS of a retroviral vector have beenaltered to remove any complementarity to the 3' CCA-tail of naturallyoccurring tRNA molecules, it may be necessary or advantageous to alterthe CCA-tail in a tRNA-like primer according to the invention to becomplementary to the altered PBS in order to optimize the system withrespect to efficiency of transfer.

Specific tRNA-like primers according to the invention, which are able toprime the reverse transcription of a retroviral vector derived frommurine leukemia virus, are derived from tRNA^(pro), tRNA^(gln1),tRNA^(lys3) or tRNA^(met)(i), in particular from tRNA^(pro).

A DNA sequence according to the invention comprises the structural genesequence of a tRNA-like primer as described above.

A suitable such DNA sequence is a synthesized oligonucleotide designedas a mutated version of the murine tRNA^(pro) gene and containing,besides the the structural sequence, a 5' leader sequence and a 3'sequence encompassing the termination signal (TTTTT), corresponding tothe flanking sequences of the murine gene; and a specific DNA sequenceof this kind has the sequence stated in SEQ ID No. 2 as illustrated inFIG. 10.

A packaging cell line according to the invention is an avian ormammalian cell line which has been transformed by the insertion of oneor more vectors comprising the DNA sequences carrying information forthe production of viral proteins required in trans for retroviralreplication, and which is characterized in that it has also beentransformed by the insertion of a vector comprising a DNA sequence asdescribed above.

A suitable packaging cell line is derived from a murine cell line.

Also, a suitable packaging cell line may be derived from any cell linesupporting MLV production. Packaging cell lines derived from non-murinecell lines may exhibit a reduced risc of recombination between vectorconstructs, packaging constructs and endogenous retroviruses.

Finally, the present invention also provides avian or mammalian cellswhich have been transfected with a retroviral vector according to theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A diagrammatic outline of a murine leukemia virus (MLV)particle.

The legend identifies individual proteins found in the mature virion.The two genomic RNAs are dimerized near the 5' end and are here shownpartly covered by NC protein. (From Whitcomb & Hughes, 1992)

FIG. 2: The genomic map of murine leukemia virus (MLV).

At the top the structure of the integrated provirus followed by the openreading frames. The provirus gives rise to two MRNA transcripts. Thefull length MRNA encodes the Gag- and the Gag-Pol polyproteins and alsoserves as the genomic RNA, while a spliced mRNA encodes the two envelopeproteins SU and TM. The subdivision of the Gag- and Gag-Pol polyproteinsinto functional domains representing the mature virion proteins is alsoshown. (Adapted from Whitcomb & Hughes, 1992)

FIG. 3: The structure of the Akv-MLV LTR.

The arrows in U3 represent the two enhancer direct repeats, IR: invertedrepeat, C: CAAT-box, T: TATA-box, p(A): polyadenylation signal.

FIG. 4: The retroviral lifecycle.

Going from left to right; a schematic outline of the lifecycle of MLV.The virus particle enters the host cell through interaction with aspecific receptor molecule. Reverse transcription is completed in thecytoplasm before the DNA/protein complex containing the viral DNAmigrates to the nucleus where the provirus is integrated into the hostgenome. Transcription of the provirus and translation of the viral mRNAis carried out by the host cell machinery. New virus particles areformed at the cell surface.

FIG. 5: A model of reverse transcription.

From top to bottom the steps leading from a single-stranded positivesense mRNA to the double-stranded DNA provirus are shown. Thin linesrepresent RNA, and thick lines represent DNA.

FIG. 6: Schematic representations of the proviral genome of MLV and of aMLV-based retroviral vector.

Upper) Division of a MLV proviral genome into sequences required in cisor in trans for retroviral replication.

Lower) The basic configuration of a retroviral vector; the gene ofinterest is inserted between the two LTRs instead of the codingsequences which are provided in trans by the packaging cell line.

FIGS. 7A and 7B: Retroviral packaging system.

7A) A retroviral packaging cell containing a simple packaging constructwhich is able to produce the viral proteins but, due to a deletion inthe packaging signal, is packaging defective.

7B) The RNA of a retroviral vector introduced into a packaging cell willbe efficiently encapsidated in recombinant viral particles.

FIG. 8: Retroviral packaging constructs.

Top: A simple packaging construct lacking the packaging signal (Ψ-2,Ψ-AM).

Bottom: An advanced packaging construct split into two separate units.The retroviral promoter-enhancer region has been substituted by aheterologous promoter (e.g. the cytomegalovirus promoter), and thepolyadenylation signal has been replaced with another (e.g. from SV 40).

FIG. 9: The basic idea of making vector transfer dependent upon thepresence of an artificial RNA primer.

Upper panel: A packaging cell without the artificial primer, from wheretransmission of the vector with the mutated primer binding site(tvAkv-XXX) is not possible.

Lower panel: A packaging cell complemented with a synthetic geneencoding an artificial RNA primer. The RNA primer (tRNA^(xxx)) matchesthe primer binding site in tvAKV-XXX and allows efficient transmissionof this vector.

FIG. 10: Complete sequence of the synthetic tRNA^(xxx) gene SEQ ID NO.2.

The gene was designed on the basis of the sequence for the mouse genefor tRNA^(pro1). The functional regions of the normal gene and theintroduced mutations are indicated. One 127 nucleotides longoligonucleotide was synthesized and made double stranded by elongationfrom an elongation primer SEQ ID NO. 25. The double stranded product wascloned into plasmid vector pUC19. Plasmid DNA was transfected into Ψ-2packaging cells.

FIG. 11: Vector recombination with endogenous MLV-like sequences.

Hypothetical model of recombination between PBS-xxx and MLV-likesequences harbouring a PBS-gln2. Recombination is most likely to occurduring minus-strand DNA synthesis. Minus strand strong stop DNA isindicated by a thick line, RNA is depicted by thin lines, and possiblerecombination-crosses are shown as dotted lines.

FIG. 12: Primer binding site (PBS) generation in rare transductionevents.

Hypothetical model explaining the rare transductions of PBS-xxx andPBS-pro.

a) Normal priming of DNA synthesis after the second strand transferevent. PBS(t) is a DNA copy of the 3' 18 nucleotides from the primermolecule. PBS(g) is the DNA copy of the genomic primer binding site.

b) Imperfect binding of tRNA primer to PBS-xxx resulting in 3' mismatchafter second strand transfer. Removal of 3' overhang by cellularexonucleases restores priming ability and regenerates PBS(g), i.e.PBS-xxx.

c) PBS-pro may be regenerated from PBS-xxx provided the copying of thetRNA primer molecule into DNA has extended beyond the methylatedadenosine residue at position 19 from the 3' end of the tRNA molecule(tRNA read-through).

DETAILED DESCRIPTION OF THE INVENTION

When testing the significance of the tRNA primer molecule in the virallife cycle of a retroviral vector in a retroviral vector propagationsystem, the effect of the introduced mutations can be quantified bymeasuring vector transduction efficiency. Furthermore, using a vectorharboring the selectable marker-gene neo (Beck et al. 1982), conferringresistance to the neomycin analog G418, individual transduction eventscan be examined by analyzing the structure of transduced provirusesafter 1 round of retroviral replication.

According to the present invention the PBS of an Akv-MVL-basedretroviral vector was modified to a sequence (PBS-xxx) that does notallow strong base-pairing with the 3' end of any known murine tRNAmolecule. PBS-xxx was designed on the basis of an alignment of thepublished murine tRNA sequences (see Table) so that the PBS-xxx sequenceis complementary to an 18 nucleotides RNA sequence showing the leastpossible homology to the 3' ends of these tRNA molecules. However, asthe transcription and maturation pathway of eukaryotic tRNA moleculesprovides all tRNAs with a 3' CCA-tail, the 5' UGG sequence of the PBSwas not modified in order to preserve complementarity with an artificialtRNA-like primer retaining the CCA-tail as described below. The chosenRNA sequence was the following: AUCCCUAGAUUCAUCCCA SEQ ID NO. 24; andaccordingly PBS-xxx has the complementary sequence stated in SEQ ID No.1 UGGGAUGAAUCUAGGGAU.

                                      TABLE                                       __________________________________________________________________________                                  GenBank/EMBL                                                  Accession                                                         tRNA3' 18 nucleotides number                                                __________________________________________________________________________    Ala (g)                                                                             UCCCCGGCAUCUCCACCA                                                                          SEQ ID NO: 3                                                                            MMTGPA1                                            - ArgCUCCUGGCUGGCUCGCCA SEQ ID NO: 4MMRL1                                     - Asp (g)UUCCCCGACGGGGAGCCA SEQ ID NO: 5MMRNO3                                - Cys (g)AUCCAGGUGCCCCCUCCA SEQ ID NO: 6MMTGCI                                - Gln-1AUCUCGGUGGGACCUCCA SEQ ID NO: 7MMTROA                                  - Gln-2AUCUCGGUGGAACCUCCA SEQ ID NO: 8.sup.a                                  - GluUUCCCGGUCAGGGAACCA SEQ ID NO: 9.sup.a                                    - Glu (g)UUCUCGGUCAGGGAACCA SEQ ID NO: 10MMRNO3                               - Gly (g)UUCCCGGCCAACGCACCA SEQ ID NO: 11MMRNO3                               - His (g)AUCCGAGUCACGGCACCA SEQ ID NO: 12MMTMT1                               - IleUCCCCGUACGGGCCACCA SEQ ID NO: 13.sup.a                                   - Lys (g)GUCCCUGUUCAGGCGCCA SEQ ID NO: 14MMRNO1                               - Lys-3GCCCCACGUUGGGCGCCA SEQ ID NO: 15MMK4                                   - Met(i)AACCAUCCUCUGCUACCA SEQ ID NO: 16MMMI                                  - MetACCUCAGAGGGGGCACCA SEQ ID NO: 17MMTRNM3                                  - MetUCCUCACACGGGGCACCA SEQ ID NO: 18MMM4                                     - PheUCCCGGGUUUCGGCACCA SEQ ID NO: 19MMF                                      - ProAUCCCGGACGAGCCCCCA SEQ ID NO: 20MMP1                                     - SerUCCACCUUUCGGGCGCCA SEQ ID NO: 21.sup.a                                   - TrpAUCACGUCGGGGUCACCA SEQ ID NO: 22TNTMS                                    - ValAACCGGGCGGAAACACCA SEQ ID NO: 23MMV1MI                                __________________________________________________________________________     .sup.a  Sequences from Sprintzl et al.  (1989). Nucleic Acids Research,       17: Supplement r1-r172.                                                       (g): Derived from sequence of tRNA gene.                                 

The mutations were introduced into the PBS of the Akv-MLV-basedretroviral vector, tvAkv-neo² (Paludan et al. 1989), using polymerasechain reaction (PCR) -mediated site-directed mutagenesis. A two-step PCRprocedure was employed for changing the PBS-pro of tvAkv-neo to PBS-xxxand to amplify the full-length transmission vector. The first stepinvolved two sub-reactions: The reaction amplifying the 5' part of thevector introduced the desired mutations and added a specific linkersequence to the upstream LTR; the reaction amplifying the 3' partcreated a small overlap with the upstream fragment and added a linkersequence to the down-stream LTR. The second PCR reaction used primersmatching the introduced linkers of the upstream and downstream LTRs toamplify the complete 3.4 kb vector. Unique EcoRI and XhoI restrictionsites present in the linkers were used for cloning of the modifiedvectors in the E. coli plasmid pUC19. The structure of the resultingplasmid clones was verified by sequence analysis of the PBS andsurrounding region and by restriction analysis. The functional state ofthe neo gene was confirmed by the ability of the plasmids to conferkanamycin resistance upon recipient bacteria. For further details on theexperimental procedure see Lund et al. 1993.

² tv: transmission vector

DNA of this vector was transferred into the Ψ-2 packaging cell line(Mann et al. 1983) and recipient packaging cells were selected by growthin geneticin (G418). Supernatant from this culture was used to infectNIH 3T3 cells and the vector titre determined from the number G418resistant colonies resulting from the infected population of NIH 3T3cells exactly as described previously (Lund et al. 1993). The titre wasreduced by a factor of 10⁵, relative to a parallel experiment with theparent vector carrying the normal PBS-sequence. The transduction titreswere 5.2×10⁶ /4.3×10⁶ for the vector with the wild type PBS and 7.6×10¹/5.4×10¹ for the PBS-xxx vector (duplicate determinations for each).

Sequence analysis of rare PBS-xxx transduction events showed that threedifferent PBSs had been transduced; PBS-xxx, PBS-pro and PBS-gln.Examination of nucleotide sequences flanking the transduced PBSsmatching a glutamine tRNA revealed a pattern of mutations indicatingthat these PBSs had originated from homologous recombination withendogenous MLV-like sequences rather than from the binding of aglutamine tRNA to PBS-xxx. Furthermore, the PBS-gln2 flanking sequencesare identical to endogenous MLV-like sequences reported by Nikbakht etal. (1985). Therefore, it can be concluded that PBSs matchingtRNA^(gln2) originate from recombination events between the introducedPBS-xxx vector and endogenous MLV-like sequences (FIG. 11). Ten of theanalyzed clones resulting from rare PBS-xxx transduction events carriedthe PBS-xxx originating from the introduced vector, and 3/26 colonieshad a PBS matching tRNA^(pro). The generation of these transduced PBSscannot be directly explained by the model for reverse transcription.However, the results can be explained asssuming weak interactionsbetween a tRNA^(pro) and PBS-xxx. PBS-xxx could be regenerated, iftRNA^(pro) primed reverse transcription from the CCA-tail only, andendogenous exonuclease activity removed the resulting 5' overhang afterthe second jump (FIG. 12b). Regeneration of PBS-pro requires erroneousread-through of the primer tRNA molecule beyond the 18th 3' nucleotidecreating a stretch of 5 nucleotides complementary to a sequenceimmediately downstream from the primer binding site. In Akv-MLV tRNAread-through creates a stretch of 5 nucleotides complementary to asequence motif downstream from the PBS. Annealing to this 3' sequence isthought to induce instability due to the number of unpaired bases, and aresulting 5' PBS mismatch may be removed by exonuclease activity leadingto the generation of PBS-pro. (FIG. 12c).

We next attempted to complement the transduction deficiency of PBS-xxxby providing Ψ-2 packaging cells with a modified tRNA that would give agood match to PBS-xxx. A gene, putatively encoding a tRNA-like primerwith a 3' end matching PBS-xxx was built from chemically synthesizedoligonucleotides. This gene ("the tRNA^(xxx) gene") was designed on thebasis of the mouse gene for tRNA^(pro1) (Russo et al. 1986) (FIG. 10) byintroduction of 18 mutations. The gene was designed to maintain thesignals for transcription and processing of this tRNA. Ψ-2 cells weretransfected with DNA for this artificial gene together with a selectablehygromycin B encoding marker gene. Cells carrying stably integratedcopies of the tRNA^(xxx) gene were generated by growth in hygromycin Bcontaining medium. These cells were transfected with DNA from thePBS-xxx vector and the titre of functional vector particles determinedunder transient expression conditions to be 2.3 10⁵ c.f.u./ml. Thistitre is in the same range as that observed under transient expressionconditions with the isogenic vector carrying the normal PBS. Usuallytransfer efficiencies observed after stable transfection of vector DNAare about 10 times higher than those observed after transient transfer,where only a fraction of the cells have received vector DNA. Additionalcharacterization of the system will involve determination of transferefficiencies after stable transfer.

4. What are the advantages of the new solution?

Our results thus indicate that impairment of the primer binding site bymutation reduces transfer efficiencies by a factor of 10⁵. However thistransfer efficiency can be restored to normal levels by complementationwith an engineered primer. This transfer principle may therefore combineefficient transfer from specifically engineered cells with severelimitations in the risk of spread under all natural conditions, where noprimer matching PBS-xxx is expected to occur. In addition it may allowvector-mediated anti-viral activitiy directed against wild-type primerbinding sites.

The technology may be further refined by alterations in the sequences ofthe modified primer binding site as well as in the artificial primer. Inparticular, changes corresponding to positions conserved in all tRNAsmay be important. The RNA primer may be expressed under control ofheterologous transcriptional regulatory elements to avoid dependenceupon the intragenic polymerase 3 promoter sequences and to optimizetranscript levels.

REFERENCES

Beck, E., E. A. Ludwig, R. Auerswald, B. Reiss, and H. Schaller (1982).Nucleotide sequence and exact location of the neomycinphosphotransferase gene from transposon Tn5. Gene 19: 327-336.

Chapman, K. B., A. S. Bystrom, and J. D. Boeke (1992). Initiatormethionine tRNA is essential for Tyl transposition in yeast. Proc. Natl.Acad. Sci. USA. 89: 3236-3240.

Cone, R. D. & R. C. Mulligan (1984). High-efficiency gene transfer intomammalian cells: generation of helper-free recombinant retrovirus withbroad mammalian host range. Proc. Natl. Acad. Sci. USA. 81: 6349-6353.

GenBank/EMBL Nucleotide Sequence Database.

Gilboa, E., S. W. Mitra, S. Goff, and D. Baltimore (1979). A detailedmodel of reverse transcription and tests of crucial aspects. Cell 18:93-100.

Lund, A. H., M. Duch, J. Lovmand, P. J.o slashed.rgensen, and F. SkouPedersen (1993). Mutated Primer Binding Sites Interacting with DifferenttRNAs Allow Efficient Murine Leukemia Virus Replication. Journal ofVirology, 67: 7125-7130.

Mann, R., R. C. Mulligan, and D. Baltimore (1983). Construction of aretrovirus packaging mutant and its use to produce helper free defectiveretroviruses. Cell 33:153-159.

Miller, A. D. (1990). Retroviral packaging cells. Human Gene Therapy, 1:5-14.

Nikbakht, K. N., C.-Y. Ou, L. R. Boone, P. L. Glover, and W. K. Yang(1985). Nucleic sequence analysis of endogenous murine leukemiavirus-related proviral clones reveals primer binding sites forglutamine-tRNA. J. Vir. 54: 889-893.

Paludan, K., H. Y. Dai, M. Duch, P. J.o slashed.rgensen, N. O.Kjeldgaard, and F. S. Pedersen (1989). Different relative expressionfrom two murine leukemia virus long terminal repeats in unintegratedtransfected DNA and in integrated retroviral vector proviruses. J. Vir.63: 5201-5207.

Russo, T., Costanzo, F., Oliva. A., Ammendola, R., Duilio, A., Esposito,F., and F. Cimino (1986). Structure and in vitro transcription of tRNAgene cluster containing the primers of MuLV reverse transcriptase. Eur.J. Biochem. 158: 437-442.

Sprintzl, M., T. Hartmann, J. Weber, J. Blank, and R. Zeidler (1989).Compilation of tRNA sequences and sequences of tRNA genes. Nucleic AcidsRes. 17: rl-rl72 (Supplement).

Valerio, D., in "Transgenic Animals", Grosveld and Kollias, Eds.,Academic Press, 1992.

Whitcomb, J. M. & S. H. Hughes (1992). Retroviral reverse transcriptionand interation: progress and problems. Ann. Rev. Cell Biol. 8: 275-306.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: RNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: AKV murin - #e leukemia virus                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..18                                                  - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1:                        - - UGGGAUGAAU CUAGGGAU             - #                  - #                      - #  18                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 127 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Mus muscu - #lus                                       - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:2:                        - - CCGGAATTCG AAAACGAAGA AACAAAGTTT ACATCTATGA ATCTGGTCTA GG -             #GGTATGAT     60                                                                 - - TCTCGCTTAG GGTGCGAGAG GTCTAGGGTT CAAATCCCTA GATTCATCAA GT -            #TTTTATAA    120                                                                 - - GCTTTCC                 - #                  - #                       - #         127                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:3:                        - - UCCCCGGCAU CUCCACCA             - #                  - #                      - #  18                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:4:                        - - CUCCUGGCUG GCUCGCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:5:                        - - UUCCCCGACG GGGAGCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:6:                        - - AUCCAGGUGC CCCCUCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:7:                        - - AUCUCGGUGG GACCUCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:8:                        - - AUCUCGGUGG AACCUCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:9:                        - - UUCCCGGUCA GGGAACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:10:                       - - UUCUCGGUCA GGGAACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:11:                       - - UUCCCGGCCA ACGCACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:12:                       - - AUCCGAGUCA CGGCACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:13:                       - - UCCCCGUACG GGCCACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:14:                       - - GUCCCUGUUC AGGCGCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:15:                       - - GCCCCACGUU GGGCGCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:16:                       - - AACCAUCCUC UGCUACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:17:                       - - ACCUCAGAGG GGGCACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:18:                       - - UCCUCACACG GGGCACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:19:                       - - UCCCGGGUUU CGGCACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:20:                       - - AUCCCGGACG AGCCCCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:21:                       - - UCCACCUUUC GGGCGCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:22:                       - - AUCACGUCGG GGUCACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:23:                       - - AACCGGGCGG AAACACCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: tRNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:24:                       - - AUCCCUAGAU UCAUCCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Mus muscu - #lus                                       - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:25:                       - - GTAGTTCAAA AATATTCGAA AGG           - #                  - #                    23                                                                    __________________________________________________________________________

We claim:
 1. A retroviral transfer vector comprising a retrovirus inwhich at least part of the genomic RNA sequences carrying informationfor the production of viral proteins required in trans for retroviralreplication have been replaced by one or more sequences carryinginformation to be introduced in a target cell chromosome, where theprimer binding site (PBS) has been modified to a sequence that does notallow strong base pairing with the 3' end of any naturally occurringtRNA, and where the three 5' nucleotides of the PBS are UGG.
 2. Aretroviral vector according to claim 1 in which the primer binding site(PBS) has been modified to a sequence showing less than 50% homologywith the 3' end in any naturally occurring tRNA.
 3. A retroviral vectoraccording to claim 1 in which the primer binding site (PBS) has beenmodified to a sequence showing less than 33% homology with the 3' end inany naturally occurring tRNA.
 4. A retroviral vector according to claim1 in which up to 5 nucleotides 3' of the primer binding site have alsobeen modified to a sequence that does not allow additional base pairingwith a naturally occurring tRNA primer.
 5. A host cell which has beentransfected with a retroviral vector according to any of claims 1-4. 6.A packaging cell line for replication of a retroviral transfer vectoraccording to any one of claims 1-4, where the cell line is a mammalianor avian cell line which has been transformed by the insertion of one ormore vectors comprising a DNA sequence carrying the information for theproduction of viral proteins required in trans for retroviralreplication, where the cell line has also been transformed by theinsertion of a vector containing a DNA sequence comprising thestructural gene sequence of a tRNA primer where said primer primes aprimer binding site (PBS) that has been modified to a sequence that doesnot allow strong base pairing with the 3' end of any naturally occurringtRNA, and where the three 5' nucleotides of the PBS are UGG.
 7. Thepackaging cell line of claim 6 where the tRNA structural gene encodestRNA^(pro), tRNA^(gln1), tRNA^(lys3), or tRNA^(met)(1) and where thesequences involved in forming the secondary clover-leaf structure of thetRNA molecule by internal base pairing with the modified sequences aremodified to be complementary therewith.
 8. The packaging cell line ofclaim 7 where the tRNA is derived from tRNA^(pro).
 9. The packaging cellline of claim 8 where the DNA sequence comprising the structural genesequence further includes a 5' leader sequence and a 3' sequence,encompassing the terminal signal TTTTT, corresponding to the flankingsequences of the murine tRNA^(pro) gene.